ABSTRACT Sedentary behavior (SB) contributes to increased type 2 diabetes risk over the lifespan. Interrupting SB is a promising strategy to reduce risk, but the long-term efficacy is not yet clear and data on the relationship between in-lab and free-living metabolic responses are lacking. The goal of this proposal is to provide training in physiologic consequences of SB, field-based assessments of glucose responses to interrupting SB via continuous glucose monitoring, and advanced multi-level statistical methods to study the moderators and the effectiveness of SB interruptions on glucose homeostasis in both in-lab and free-living contexts, yielding data important for the development of targeted type 2 diabetes prevention intervention strategies. Mentored training will consist of: (1) training in expertise in longitudinal study design and field-based data collection techniques in youth; 2) the pathophysiology of type 2 diabetes; 3) the use of advanced statistical modeling techniques to analyze longitudinal variations in energy balance behaviors as they relate to metabolic outcomes; and 4) the design of field-based intervention strategies to improve and address type 2 diabetes-related physiologic mechanisms. Dr. Michael Goral (co-primary mentor) is an expert in the causes and consequences of childhood obesity and type 2 diabetes risk factors in youth. Dr. Genevieve Dunton (co-primary mentor) is an expert in field-based energy balance behavior assessment and longitudinal study methodology. Drs. Jimi Huh and Kathleen Page (co-mentors) are experts in statistical models for health behavior research, and endocrinology and type 2 diabetes pathogenesis. Specific Aim 1 tests the hypothesis that individual-level factors modify in- lab and free-living metabolic response to experimental conditions. Specific Aim 2 tests the hypothesis that the magnitude of in-lab glucose responses to SB interruptions will be associated with the magnitude of free-living glucose responses to SB interruptions over 4 days in a subsample (N= 12). Participants (28 normal weight, 28 overweight/obese; ages 11-15) from an ongoing observational study (N=202) will complete an in-lab randomized crossover trial of acute metabolic responses to prolonged vs. SB interruptions; and a subset (6 healthy weight, 6 overweight/obese) will complete two 4-day study of effects of free-living habitual activity vs. device-prompted SB interruptions on glucose homeostasis. In-lab studies can help elucidate physiologic mechanisms of SB interruptions, but are limited in their non-naturalistic and highly controlled nature. Understanding the longer-term efficacy and physiologic consequences of interrupting SB is essential for the design of effective intervention strategies to prevent type 2 diabetes in youth.